The Unit of Molecular Signal Transduction, directed by Tamas Balla, investigates signal transduction pathways that mediate the actions of hormones and growth factors in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Current studies are aimed at (1) understanding the function and regulation of two novel phosphatidylinositol (PI) 4-kinases, PI4KIII-alpha and PI4KIII-beta; (2) characterizing the structural features that determine the catalytic specificity of PI 3- and PI 4-kinases; (3) defining the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology domains of selected regulatory proteins; (4) determining the importance of these interactions in the activation of cellular responses by G protein- coupled receptors and receptor tyrosine kinases.The expression pattern of the two distinct type III PI 4-kinases was compared by in situ hybridization histochemistry in the rat using 35-S-labeled antisense riboprobes specific for the two sequences. PI4KIII-alpha was found to be predominantly expressed in the brain, with low expression in peripheral tissues. In contrast, PI4KIII-beta was more uniformly expressed, and in addition to the brain was present in several peripheral tissues including the kidney, lung, spleen, heart, and the seminiferous tubules of the testis. Moreover, within the brain, PI4KIII-beta showed a more uniform expression pattern, the most pronounced signal being found in the gray matter, especially in neurons of the olfactory bulb and the hippocampus. While these structures also showed a strong signal with PI4KIII- alpha, the latter was not detectable in the white matter and in the choroid plexus, both of which were positive for PI4KIII-beta. In the retina, both enzymes were expressed in the pigment layer and in both nuclear layers, and the ganglion cells. In a 17 day old rat fetus, PI4KIII-beta was more widely distributed and PI4KIII-alpha was primarily expressed in neurons. These results indicate that both PtdIns 4-kinases are widely expressed and probably co-exist in many cells, although in different proportions. This expression pattern, and the conservation of these two proteins during evolution, suggest that they have non-redundant functions in mammalian cells. In an attempt to better characterize PI4KIII-beta, a GST-PI4K-beta fusion protein was created for expression in E. Coli. Using mild inducing conditions in the bacterial strain BL21, it was possible to obtain a soluble protein which was then purified by GST-agarose chromatography and cleaved with PreScission protease. The purified protein was biologically active and phosphorylated PI in its 4- position, with wortmannin-sensitivity and kinetic parameters that were identical to those of the purified bovine brain PI4KIII-beta. In addition to its lipid kinase activity, the enzyme was found to undergo autophosphorylation, a feature that was enhanced by Mn2+ ions, and which was also inhibited by wortmannin and another PI 3- kinase inhibitor, LY 294002. The recombinant protein was unable to transphosphorylate, indicating that the autophosphorylation site, which was located to the C-terminal catalytic domain of the protein, is held in position by intramolecular interactions. Autophosphorylation was found to inhibit subsequent lipid kinase activity, suggesting that it may represent a regulatory mechanism for the enzyme. This is the first PI kinase that has been expressed in a functional form in prokaryotes. Identification of the intracellular membrane compartments where inositol lipids are synthesized in response to stimulation would greatly enhance our understanding of the manner in which these lipids regulate signaling processes within the cell. In an attempt to image the lipid product of PI 3-kinases within live cells, the pleckstrin homology (PH) domain of the Bruton?s tyrosine kinase (Btk), which has been reported to bind 3-phosphorylated inositides, namely PI(3,4,5)P3 in vitro, was fused to the enhanced green fluorescent protein (EGFP). This construct was expressed in NIH 3T3 cells and its distribution examined by confocal laser microscopy. Incells that were rendered quiescent by serum-deprivation, the distribution of the fusion protein was indistinguishable from that of EGFP alone. However, after stimulationwith PDGF the fusion proteinwas translocated from the cytosol to theplasma membrane, and this process was rapidly reversed by concentrations of wortmannin thatspecifically block PI3-kinases. Similar translocation was observed using other cells and stimuli that activate PI 3-kinases. The R28C mutation within the Btk PH-domain that causes the human disease, X-linked agammaglobulinemia, completely prevented the localization of the BtkPH-EGFP protein to membranes after stimulation, consistent with previous reports that this mutation interferes with the membrane targeting of the kinase. Another mutation within the PH domain of Btk (E41K), which renders Btk a transforming kinase in NIH 3T3 cells, was found to cause localization of the fusion protein to the plasma membrane even without stimulation. These experiments demonstrated that the PH domain of Btk is sufficient to localize the protein to the plasma membrane in a PI 3-kinase-dependent manner, and could be used to visualize changes in PI(3,4,5)P3 levels in single living cells. These data also clearly demonstrated the biochemical consequences of a single mutation that causes a human disease through defective or altered localization of a protein at the cell membrane level.